The Nitrogen Cycle - The Paradigm Shifts

In an earlier posted article, Archaea The not-so-new-kids-on-the-block, we briefly discussed the emergence of certain species of the Domain Archaea as the prime ammonia oxidizing organisms in both terrestrial and aquatic environments world-wide.
Over the past several years there has been increasing evidence that a similar altering of perceived role dominance is extant in Nitrite oxidation.
Some bacterial and archaeal species defy replication under lab condition and have also defied easy identification by past known means. The bacterium Nitrospira is one such micro-organism. Only recently has a method been developed allowing for somewhat better identification. As a result, what has been discovered has changed the paradigm not only as relates to Nitrite oxidation but also, to a point, Ammonia oxidation.
Enough evidence has been amassed to date to confirm that in many aquatic environments Nitrospira is the dominant Nitrite oxidizing specie instead of Nitrobacter. This is most noticeable in waters that have an elevated Ammonia level. These elevated Ammonia levels inhibit the Nitrite oxidizing process in Nitrobacter but has no effect on the efficiency of Nitrospira. This means in an aquatic habitat where the Nitrogen cycle is not established or balanced Nitrospira will likely be the dominant Nitrite oxidizer with Nitrobacter increasing in numbers only when Ammonia levels have been reduced. This does not mean that Nitrospira’s role becomes diminished. Nitrospira has the almost unique ability to also convert Urea into Ammonia. It forms a quasi-symbiotic relationship with Nitrosomonas and, it is suspected, Archaea, supplying Ammonia in ezchange for Nitrite. So even in an established well-balanced aquatic habitat the Nitrite oxidizing duties are at least shared by Nitrobacter and Nitrospira.
In addition, Nitrospira remain active in anoxic conditions.
No longer are certain processes relegated exclusively to certain micro-organisms, but rather new organisms have emerged as the dominant players that are capable of performing multiple processes in the Nitrogen cycle.
The paradigm continues to shift.

Archaea--Update

In 1977, while Dr. Carl Woese and his colleagues at the University of Illinois were using a then new process of DNA sequencing for studying relationships between bacteria, it was discovered that there were two distinctly different groups. Those “bacteria” that lived at high temperatures (extremophiles) or produced methane formed a group quite different genetically from the usual bacteria. Because of this vast genentic difference in makeup, Dr. Woese proposed that a new domain of life be added…Archaea.

The scientific community was understandable shocked by this proposal and for several years balked at accepting such a major revision in century old thinking. Further research subsequently validated Dr. Woese’s proposal and in 1990 the existing three domains of life (Bacteria, Archaea, Prokaryote) were created.

Initially thought to exist only in extreme environments, those devoid of oxygen and whose temperatures were near or above the boiling point of water, microbiologists soon realized that Archaea are a large and diverse group of organisms that are ubiquitous to all environments-terrestrial and aquatic and significant contributors to the global carbon and nitrogen cycles. It is the role of Archaea in the nitrogen cycle (specifically aquatic) that is of interest and the focus of this document.

Since the acceptance of Archaea as a separate Domain of life, research has been both intensive and massive, delving into all aspects of this life form. Much of this research has been on the role of Archaea in the Nitrogen Cycle with the results of this research simultaneously upending some long held beliefs and yet clarifying other processes.

For Pondkeepers, maintaining the equilibrium of the Nitrogen cycle is of the first order of importance. To be able to do so requires a certain level of knowledge of how the involved biological processes work. Most Pondkeepers know that Ammonia is oxidized by Nitrosomonas bacteria et.al. to Nitrite which is oxidized by Nitrobacter bacteria et.al. to Nitrate which is assimilated by algae and plants or reduced to Nitrogen gas by other bacteria. Archaea do nothing to change this, but supplement the process, providing, shall we say, more efficiency in the initial Ammonia oxidation stage.

AOA (Ammonia Oxidizing Archaea) and AOB (Ammonia Oxidizing Bacteria) both occupy important niches in the Nitrogen cycle. In Oligotrophic (low nutrient) waters, AOA are the predominant organisms and in Eutrophic (high nutrient) waters AOB dominate.

In the world’s oceans, AOA are now known to be the primary oxidizers of Ammonia, replacing AOB which for decades were believed to have performed this function. The same has been found true in many freshwater lakes and even in some soils. So what does this mean to a Pondkeeper?

Although no true research has been undertaken targeting ponds specifically, there have been two (2) papers^1,2 (2011 and 2014) addressing the roles of AOA and AOB in aquaria. Additionally,  a Master’s thesis³ on this subject was also written in 2014. The results of these three (3) documents can logically be applied to garden ponds as both (ponds and aquaria) are closed systems.

What was revealed in all of these research documents was that in established and balanced aquaria, AOA were not only the dominant, but in some cases, the only oxidizer present. This is not to imply that AOB have lost their status as being an integral part of a pond’s Nitrogen cycle, but that their role of importance is limited to establishing the initial balance in a new pond when Ammonia levels are high.

This begs the question: Does this really change anything?

The simple answer is…No. It does, however, because of some unusual characteristics of AOA, offer a different perspective on the Nitrogen cycle process.

For instance, it has been shown that AOA are fully capable of Ammonia oxidation in suboxic (low oxygen) conditions. They have been isolated from the sludge at various Waste Water Treatment Plants which were almost devoid of Oxygen. How Archaea are able to do this is still being researched.

It has also been suggested by some, denied by others, that AOA are still able to function at temperature approaching freezing. If proven true it could make a difference to Pondkeepers in higher Latitudes.

As yet, Archaea have only been found that convert Ammonia. None have been found that oxidize Nitrite. Considering the fact that research into this amazing organism is really in its infancy, future surprising discoveries can certainly not be ruled out.

For certain, with the newly acquired knowledge of AOA and Anamox (a subject for another discussion), a new paradigm is required for the Nitrogen Cycle.

References:

1. Temporal and Spatial Stability of Ammonia-Oxidizing Archaea and Bacteria in   Aquarium Biofilters

     Samik Bagchi1.¤, Siegfried E. Vlaeminck1., Laura A. Sauder2, Mariela Mosquera1,

Josh D. Neufeld2, Nico Boon1

2.  Aquarium nitrification revisited: thaumarchaeota are the dominant ammonia oxidizers in freshwater aquarium biofilters

     Laura A. Sauder, Katja Engel, Jennifer C. Stearns¤, Andre P. Masella, Richard Pawliszyn, Josh D. Neufeld

3. Ecology of Ammonia-oxidizing Archaea and Bacteria in Freshwater Biofilters

     Natasha Alexandria Szabolcs

Periphyton Part 2 - Aufwuchs

Periphyton

Part 2

Aufwuchs

In a previous article, we discussed the formation processes and structure of biofilm in aquatic environments. We now turn to aufwuchs….its formation and composition, and the functions of the member flora and fauna.

As biofilm approaches its final stages of growth, which is determined by both organic and inorganic nutrients, algae begins to colonize the biofilm surface. Diatoms are usually first to colonize but are quickly followed by other sessile forms of algae including filamentous. The biofilm and algae form a mutually beneficial partnership. The biofilm supplies nutrients to the algae and the algae in turn supplies much needed Oxygen to the biofilm bacteria.

Earliest algal colonization cells excrete mucilage then organized into pads, stalks with contribution of other organisms (Goldsborough, 2005). There are different adaptations changing with different taxonomic composition such as stalks with sticky ends, sticky capsules, muscular suction pads, glue or simply clinging to the substrate (Reid and Wood, 1976) later, chemical and physical factors develop the assemblage and make it denser.

Two of the factors that influence the algae growth and colony population are light and temperature.

Light had no important effect on abundance of algae, different light intensities form different algae taxa in aufwuchs structure (Vermaat J. E., 1995). For example if sufficient light is available, the community will be microalgae dominated. If there is light penetration, the aufwuchs community will be heterotroph dominated (Goldsborough, 1993). Although in very low light regimes (12 μmol) algae growth is minimal (Hill and Fanta, 2008), aufwuchs structure can support high irradiance (800 μmol) exposure

(Nofdianto, 2010). Photosynthesis occurs at levels far below maximum daily irradiance and photoinhibition is typically rare (Goldsborough et al., 2005). Temperature has effects on aufwuchs taxonomic structure like light regime (Vermaat J. E., 2005). For example, while high temperatures make the algae Scenedesmus dominate, low temperatures favored the algae Navicula in Vermmat and Hootsmans (1994)’ and Bothwell’s (1988) study. Besides it is same for season differences that diatoms are dominant in spring and green algae or cyanobacteria are dominant in summer (Meulemans and Roos, 1985).

Temperature and light often show strong parallelism because if there is sunlight, it will provide light for phytosynthesis of macrophytes and warm up the environment (Vermaat J. E., 2005). Therefore separation of temperature and light interaction and impacts on algae growth is not very common (Bothwell, 1988). Still there are some studies (Vermaat and Hootsmans, 1994; Nofdianto, 2010) which showed the interaction of low temperature and high irradiance or vice versa. Both of these studies determined the maximum growth at 20°C and 200-225 μmol. This degree of temperature and light is optimum for all organisms groups. Above or below these levels taxa and abundance is changing.

Nutrient effect on algae development has been studied many times. These studies (Marcus, 1980; Hansson, 1992; Jones and Sayer, 2003;  Liboriussen et al., 2005, 2006; Smith and Lee, 2006; Becares et al., 2008; Özkan et al., 2010; Rosemond et al., 1993, 2000; Chételat et al., 1999; Sharifi and Ghafori, 2005; Greenwood and Rosemond, 2005; Bowes et al., 2010) generally concluded that higher nutrient caused higher algal biomass in aquatic ecosystems.

Substrates greatly influence aufwuchs population diversity and richness. Sand and silt provide the poorest habitat, because of few attachment sites for algae and consumers.  Boulders, rocks and gravel are better because of larger surface area, more attachment sites, providing cover and refuge, and divert the force of moving water. Organic substrates such as logs, stumps, and tree branches are known to provide the best substrate for supporting taxa richness because of the immediate availability of carbon and other organic compounds. Twigs and leaf litter also support an abundant community but are not long-lived in the aquatic environment thus providing only temporary benefit. Aufwuchs are classified by substrate (all of the epi’s mentioned in Part 1 of this article) and by certain water quality parameters (pH range, nutrient levels of N and P, the Halobian spectrum - chloride concentrations or conductivity, and the Saprobien system - based on organic pollution).

It is a well known fact that diversity in any ecosystem is the ultimate key to the stability of that ecosystem. This especially holds true within aquatic ecosystems. Mixed populations have the advantage of being able to perform more complex transformations (for example, those requiring multiple steps), and they are more resistant to environmental stress. (Brenner, K., You, L. & Arnold, F. H.)  The more diversity and abundance an aufwuchs algae population can sustain the more diverse and abundant will be the population of the various micro-, meio-, and macro-organisms within the aufwuchs. In addition, the potential for diversity also increases the community’s ability in remediating pollution, organic and inorganic. The types of algae within an aufwuchs community are also indicators of, not only form but also, level of pollution. In a typical garden pond N and P are usually the two pollutants of concern; however other pollutants may be encountered, such as heavy metals, herbicides and pesticides.

Living upon, and within, the diverse aufwuchs algae colony is a variety of animal life ranging in size from the micro- (amoeba, protists, ciliates, flagellates etc.) through the meio- (nematodes, rotifers, harpacticoid, cyclopoid and calanoid copepods, ostracods, oligochaetes, tardigrades and gastrotrichs. Nemotodes being most populous). Meiofaunal organisms play an important role in the aquatic food web complex since they consume bacteria, other microfauna and flora, and detritus, and they are, in turn, consumed by many larger macrofaunal invertebrates and juvenile finfish (Stickney et al. 1975, Bell and Coull 1978, Alheit and Scheibel 1982, Kennish 1986, Smith and Coull 1987, Coull 1990), and macro-organisms (Insect larvae, nymphs, and adults; crustacean, amphibian larvae and fish larvae). Each, to some degree, relies on the aufwuchs as a source of food. These organisms are classified by method of feeding: raspers, grazers, scrapers, piercers and gatherers. Larger organisms such as herbivore and omnivore fish also look to aufwuchs as a source for food. Nemotodes, copepods and turbellarians comprise more than 95% of the meiofauna in most aufwuchs.

Since aufwuchs is sedentary, it is sensitive to changes in water quality. A diverse community can be found in healthy streams and ponds. Nuisance blooms are usually symptoms of a system stressed by factors such as elevated nutrients and high temperatures.

In conclusion, the periphyton community, biofilm and aufwuchs, can be considered to be the recycling center for any aquatic eco-system. It is here that the food chain begins. It is here that organics and inorganics are converted to other forms that serve as nutrients for various organisms, both small and large. It is here that feces from the various inhabitants of the ecosystem are turned back into a nutrient source. It is here that nitrification (and denitrification) occurs. It is here that inorganic pollutants such as heavy metals and chemicals may be rendered less harmful through biological processes and absorption. It is here that a large portion of total water column oxygenation takes place. It is here that the greatest mechanisms and processes for maintaining water quality exist. 

It is usually green. It is almost always slimy. It is seldom attractive. It is universally cursed and derided by many Pond Keepers. It is, however, the most important grouping of organisms in any aquatic eco-system. It is generally called Periphyton.

Pond Overstocked?

Warning: This is quite lengthy for a blog post.

Throughout the long history of decorative fish keeping, one of the many diverse problems that have been encountered is overstocking…just too many fish.

There have been countless articles written on this subject with the various pronouncements of how many fish are too many as diverse as the menu choices in a family-style restaurant.

The rapid growth of the Aquaculture industry has spurred intense scientific research into fish husbandry and all of its facets. The aim of this research has been focused on maximizing production of a quality product, much like any other industry. In Aquaculture, this translates into producing an optimum number of the largest and healthiest fish in the least amount of space.

We will examine the results of this research into optimum fish loads later in this article, but first we will examine how overstocking affects various water quality parameters and fish physiology.

In this article, decorative ponds will be considered comparable to stocking or holding ponds and tanks that are used in aquaculture.  Decorative ponds in many instances are smaller in capacity than most aquaculture  RAS holding/storage structures. It is logical, therefore, that any adverse conditions that may occur in aquaculture would certainly be duplicated or possibly magnified in a decorative pond.

Stress is defined as a condition where a fish’s internal homeostasis is impaired from a stressor (Wendelaar Bonga 1997; Barton et al. 2002).

Water quality is one of the most important contributors to fish health and stress level. Fish may be able to tolerate adverse water quality conditions; however, when adverse conditions occur from a concert of environmental and biological variables, fish may be quickly overcome by the resulting physiological challenges. The effects of water quality vary considerably with species, life stage, and previous exposure to stress (Wedemeyer 1996)

Crowding (i.e., nearing or in excess of the maximum density of fishes in a confined space) depends on a fish’s behavioral and physiological requirements for space as the carrying capacity for a given volume of water is approached. Carrying capacity is greatly governed by water quality and when it is approached or exceeded, may contribute to lowered metabolism, immunosuppression, increased disease transmission, and lowered survival rates. ( Portz , Woodley, Cech Jr. 2006)

We know that water quality is largely determined by the atmosphere, various sediments, organisms and their waste, and the flow rate in a pond. It can also be affected by the condition of the pond itself. . Fishes may incur additional energetic costs associated with stress responses from physical and chemical fluctuations in aquatic systems (Barton and Iwama 1991). Poor water quality can prompt the reallocation of energy from secondary (non-essential) physiological processes (e.g., growth, reproduction) towards primary (essential) processes (e.g., metabolism, immune function). Thus, adequate, or preferably ‘‘optimal’’, water quality is essential for holding fish in an environment that will neither activate their stress responses nor alter their normal energy budget. Exposure to poor water quality can result in permanent damage or mortality if physical or chemical variables are allowed to reach lethal levels and/or synergize in a deleterious manner (Carmichael et al. 1984a; Robertson et al. 1987; Erickson et al. 1997; Pavlidis et al. 2003;  Portz , Woodley, Cech Jr. 2006).

Flow rate - the ability of the pond to support a fish density largely depends on the water flow rate and the filtration system needed to maintain the optimal water quality required for the species.

Loading density -  refers to the actual fish carrying capacity of the water and is calculated as fish weight per water flow rate, which is the volume of water turnover in a given amount of time (i.e., kg/ l/min or lb/gpm flow).

Crowding or confinement -  refers to fish behavioral responses to the density of fishes in a given volume (Wedemeyer 1996; Timmons et al. 2002). The loading density for any given pond changes with the species, average size of fish, and water quality variables (Haskel 1941; Westers and Pratt 1977; Avault 1996; Timmons et al. 2002).  High fish loading densities can rapidly deplete DO, particularly if the fish are allowed to forage and are hyperactive or stressed. Also, fishes kept at high densities frequently incur more physical abrasions with loss of scales and mucus leading to decreased immunocompetence and increased disease persistence (Pickering and Pottinger 1989; Schreck 1996; Iguchi et al. 2003). Fish respiration enhanced by crowding reduces DO and increases dissolved CO2 concentrations, while metabolism-related secretions increase the ammonia (NH3), total suspended solids (TSS) and total dissolved solids (TDS). Assuming the temperature remains the same, pH will decline with the increasing dissolved CO2; while low pH will decrease the proportion of toxic NH3, it favors increase of dissolved CO2 (Randall 1991).

Flow rates (l/h) within a pond should be sufficient to promote optimum water quality and the removal of fish wastes, without causing fish to become stressed and physically overwhelmed by the fast flows (Burrows and Chenoweth 1970; Portz , Woodley, Cech Jr. 2006).

Temperature -  affects the physical and chemical properties of water and all chemical reaction equilibria. Typically, a 10C increase in water temperature results in the doubling of a chemical reaction rate (Stumm and Morgan 1996). Water temperature influences gas solubility, and, therefore, the dissolved gas content in water. For example, as water temperature increases, the solubility of oxygen (O2) and other gases decrease (i.e., N2 and CO2; Colt 1984). Temperature directly affects the H+ activity and must be taken into account when determining and recording pH, the carbonate cycle equilibrium and the proportion of toxic NH3 present. The carbonate cycle regulates pH, which in turn affects the NH3–NH4 + equilibria (Stumm and Morgan 1996). Water temperature also affects the lethality of pollutants on fish. For example, fish exposed to pollutants typically have a decreased survival time by a factor of two or three with each increase of 10C (Alabaster and Lloyd 1982; Heath 1998; Portz , Woodley, Cech Jr. 2006).

Effects of temperature - Fish are poikilotherms, so their surrounding water temperature is critical to their physiological reaction rates and metabolic processes. Every species has an optimal temperature range in which the fish does not exhibit any signs of thermal stress and/or altered behavior. As a fish’s body temperature increases, biochemical reaction rates generally increase for enzyme reactions and membrane transport flux dynamics, even relative to minute temperature shifts of less than 0.5_C (Elliot 1981; Neill and Bryan 1991). Consequently, a rise in water temperature often drives increased metabolic rates of fishes, assuming that there are no other limiting factors.

Thermal stress occurs when the water temperature exceeds the optimal temperature range, thus initiating changes that disturb normal physiological functions resulting in energy expended towards stress responses, and even a potential decrease in individual survivorship (Brett 1958; Fry 1971; Elliot 1981). Optimal temperature ranges may change with ontogeny, body size (Brett 1956; Fry 1971), and the individual’s health, including past thermal experiences (Elliot 1981). For example, most fishes can acclimate to gradual temperature changes over months, such as with season. However, rapid water temperature changes or exposures to sustained temperatures outside the optimal range (thus, sub-optimal) often result in thermal stresses or lethal conditions. The severity of the thermal stress response may depend on other water quality constituents, for example, DO content and TDS concentration in the water (Hettler 1976; Claireaux et al. 1995; Claireaux and Audet 2000). Thermal stress-related behaviors include a reluctance to feed, sudden or erratic movements with possible collision with the pond wall or other fishes, jumping, rolling, pitching, color and parental care change, and increased regurgitation, defecation and gill ventilation (Elliot 1981; Alsop et al. 1999; Cooke et al. 2003a; Smith and Hubert 2003; Portz , Woodley, Cech Jr. 2006).

Dissolved oxygen - The oxygen concentration in water decreases with increasing water temperature, salinity, and decreasing atmospheric pressure. The freshwater DO concentration is the greatest at 14.60 mg/l (equivalent to ‘‘parts per million’’, ppm) at air saturation and 0_C, then declines to 9.26 mg/l at 19_C, and to 7.54 mg/l at 30_C (Colt 1984) due to decreased gas solubility. At high elevations, the atmospheric pressure declines, decreasing the partial pressures of all gases, including that of oxygen (denoted as pO2). Thus, even though oxygen remains 20.94% by volume of air, the ‘‘thinner’’ air (decreased barometric pressure) decreases pO2, and consequently the maximum DO content at air equilibrium. For example, at 19_C, the expected air-equilibrated freshwater DO content decreases from 9.26 mg/l at sea level  to 8.42 mg/l at 8,000-m elevation, and to 7.65 mg/l at 1,600-m(?) elevation (Colt 1984). Conversely, water may become supersaturated (i.e., when a gas concentration is greater than that with air saturation) with DO when water is under pressure from deep wells or aquacultural technology (e.g., U-tubes), turbulently mixed in plunge pools below water falls or dams, or through intense photosynthesis in shallow pools with dense macrophytes.

Daily fluctuations and rapid depletions of DO can occur: (1) if algae is present, (2) when the loading densities are excessive, (3) as water temperature, TDS or salinity increases (Stecyk and Farrell 2002),  and (4) where fish are hyperactive and/or stressed (Wilson et al. 1994; Carey and McCormick 1998; Portz , Woodley, Cech Jr. 2006).

TAN – In instances of possible overcrowding, Tan, NO2, and NO3 should be closely monitored. . Percentage of free NH3 can be determined from water pH, temperature, and TAN. Ion exchange minerals (i.e. zeolites) may be utilized for TAN reduction, but this may be ineffective in ponds that maintain salinity levels

pH – Where  fish density is high, pH should be closely monitored. High CO2 levels will result in a decrease in pH. At high pH levels, the toxic form of ammonia will become more prevalent. The microbes involved in biological filtration consume both DO and alkalinity. The addition of bases (such as NaHCO3) to maintain a level of alkalinity is often recommended. Such additions (buffering) will also prevent the system’s pH from declining to levels that would impact both the biological filtration and, potentially, the fish.

CO2 - A recommended maximum dissolved CO2 concentration range is 15–20 mg/l, though it is not strongly supported by research (Smart 1981; Timmons et al. 2002).

Alkalinity - Some alkalinity (> 50 mg/L) is essential for maintaining optimal pH and supporting biological filters

Stocking Density - It is important to note the difference between loading and density when addressing carrying capacity for ponds. Loading is defined as the weight of fish per unit of flow (kg/l/min), while density refers to the weight of fish per unit space (kg/m3).

High densities contribute to deteriorating water quality when being held in ponds with inadequate filtration or water flow ( Pickering and Stewart 1984; Wedemeyer et al. 1990; Kebus et al. 1992). High fish densities can lead to hypoxia and accumulation of ammonia, which can be detrimental to fish health.

However, fish biomass may be just one measure of fish density for predicting water quality changes or stress effects. For example, does one large fish have the same effects as multiple smaller fish of the same total biomass? (Portz , Woodley, Cech Jr. 2006).

High stocking density impacts metabolism and growth, most often attributed to decreased food consumption and conversion (Refstie 1977; Vijayan et al. 1990; Alana¨ra¨ and Bra¨nna¨ s 1996), agonistic social interactions Fenderson and Carpenter 1971; Refstie and Kittelsen 1976; Fleming and Johansen 1984; Brown et al. 1992; Wedemeyer 1996; 1997), or diminished water quality (Pickering and Stewart 1984; Kwak and Henry 1995; Plumb et al. 1988). This expenditure of a fish’s energy reserves is exacerbated by reduced food consumption (Refstie 1977; Vijayan et al. 1990; Alana¨ra¨ and Bra¨nna¨ s 1996). Vijayan et al. (1990) suggest that food consumption decreases, as a response to a stressor (e.g., high stocking density), may be an adaptation by which stored body reserves are directed for utilization in maintenance functions (Portz , Woodley, Cech Jr. 2006).

Crowded fishes have been shown to increase mobilization of triglycerides to meet metabolic demand by improving the gluconeogenic capacity from glycerol (Vijayan et al. 1990; Montero et al. 1999). Liver weight and the hepatosomatic index are thus significantly reduced due to higher hepatic lipid utilization (Leatherland and Cho 1985; Papoutsoglou et al. 1987; Vijayan et al. 1990; Montero et al. 1999; Barton et al. 2002). At the same time, liver 3-hydroxylacyl CoA dehydrogenase, an enzyme implicated in ßoxidation, is elevated (Vijayan et al. 1990). Fatty acid composition of the liver is altered due to highstocking density, which can be observed by the reduction in oleic acid. Oleic acid is one of the principal lipid energy sources when fishes are under high energy demand (Montero et al. 1999). Another principle energy source is glycerol, which is mainly metabolized in the liver. Elevations of glycerol metabolizing enzymes (i.e., glycerol kinase, glycerol-3-phosphate dehydrogenase) of fishes held at high density suggest that this condition enhances the gluconeogenic process from glycerol (Vijayan et al. 1990). These precursors are utilized to make glycogen for storage in the liver; however, the increased maintenance requirements of a fish under stress exceeds the rate of glycogen synthesis, resulting in a glycogen debt. Thyroid hormones, which promote protein synthesis (i.e., anabolic processes), are at reduced levels in the plasma of fishes held at high densities (i.e., thyroxin [T4]; Vijayan et al. 1990). This suggests a density-dependent inhibition to thyroxin production which may result from decreased food consumption. (Portz , Woodley, Cech Jr. 2006).

Investigations of metabolic rates assessed by cardiac responses during live-well retention under different stocking densities have revealed that as density increased, cardiac output and heart rate also increased while stroke volume remained the same (Cooke et al. 2002). Another interesting finding from the Cooke et al. (2002) study is that the addition of salt and water conditioners more than doubled the time required for cardiac parameters to normalize. These findings point out an interesting contradiction with others that have found stress to be minimized by the addition of salt, water conditioners, and antibacterial agents (Carmichael et al. 1984b; Plumb et al. 1988; Swanson et al. 1996; Portz , Woodley, Cech Jr. 2006).

Health and immune function - Fish immune function responses to stress indicate that the type, intensity, and duration of the stressor are major determinants of the immune response. Improved fish immune function (i.e., increased concentrations of lysozymes, cytotoxic cells, and phagocytes; Demers and Bayne 1997; Ruis and Bayne 1997; Ortun˜o et al. 2001) has been observed following an acute stress. In these cases, the fish’s body mobilizes the immune system after acute stress, presumably protecting itself from potential subsequent trauma. Most often, deleterious effects associated with immune system dysfunctions result from both severe acute and chronic stress (Yin et al. 1995; Ortun˜o et al. 2001; Vazzana et al. 2002). Elevated (especially, prolonged) plasma cortisol levels act as an immunosuppressant in fish (Pickering and Pottinger 1989; Barton and Iwama 1991; Montero et al. 1999; Vazzana et al. 2002; Portz , Woodley, Cech Jr. 2006).

Mortality at high densities has been commonly ascribed to poor water quality, predation/cannibalism, and disease. Poor water quality can place an extra burden on an already physiologically compromised fish under high densities. Dissolved oxygen concentration may be low or toxic compound (e.g., ammonia) concentrations may be high in the water. The risk of pathogen transmission in high stocking-density systems is elevated because of the closer proximity of fish (Bosakowski and Wagner 1994a; Wedemeyer 1996; Portz , Woodley, Cech Jr. 2006).

Most often, mortality of fishes held at high densities is not the result of a single stressor. The cumulative and physiologically interactive effects of sublethal stresses that occur may be lethal, even if each independently does not exceed a physiological threshold (Carmichael et al. 1984b; Barton et al. 1986; Portz , Woodley, Cech Jr. 2006).

As we can see, many factors are involved in maintaining homeostasis in Pond fish. A more detailed, scientific description has been presented of the effects of overcrowding because it is important to know how various physiological aspects of the fish such as organ function and enzyme levels are affected. The observance of these various physiological manifestations serve as an alert to a developing or existing problem with overcrowding.

The optimum flow rate is generally agreed upon as no less than what is required to affect a complete water changeover every 30 minutes regardless of density.

We have now come to the question - at what fish density does the term ‘overcrowding’ apply?

 The simple answer is that there is no simple answer. There are, however, general guidelines that have been developed through research and in field observations. All of these guidelines assume adequate bioconversion and filtration. Many are not specie specific.

The following chart is such a guideline.

NOTE: 1 cubic meter = 265 gallons

Although this chart only shows a maximum 10 day period, it is obvious that the longer fish are held under conditions A, B, or C the less total weight of fish can be safely held.

(Enache, Cristea, et.al.) showed that common carp (Cyprinus carpio) showed a better growth rate and food conversion efficiency at 32 kg/m3 than at 64 kg/m3. (Costa-Pierce, Soemarwoto) determined that the optimal stocking density for highest growth for common carp (Cyprinus carpio) was 6.0 kg/m3 or 1.0 lb/ft3 or 1 lb/20 gallons of water or one 12 inch Koi per 20 gallons.

So it appears that Koi can tolerate quite high physical crowding assuming that water quality is optimum. The key controlling factor of determining maximum fish or stocking density of a pond is water quality. Water quality is determined by the level of bioconversion available, As stocking densities rise, then also must the level of bioconversion or bio-filtration. We can deduce from this that the level of bioconversion or bio-filtration available MUST be determined by the fish density and NOT by the water volume of a pond.